Abundant evidence indicates that carotenoids, which are present in many fruits and vegetables, decrease the risk of some cancers in humans. The provitamin A carotenoid beta-carotene inhibits neoplastic development in several cellular and animal models of cancer, apparently by a mechanism that does not involve its conversion to vitamin A. beta-Carotene and other carotenoids are postulated to inhibit carcinogenesis by acting as antioxidants, which scavenge reactive free radicals, singlet oxygen and other oxidants involved in carcinogenic tissue injury. The overall objective of the proposed project is to test this "antioxidant hypothesis" by measuring antioxidant actions of beta-carotene in cell models where beta-carotene inhibits carcinogenesis. The initial objective of the project is to identify products formed by free radical scavenging and singlet oxygen quenching reactions of beta-carotene. Both chemically defined model systems and biological membrane systems will be used to identify beta-carotene oxidation products, which will be isolated by high performance liquid chromatography and characterized by UV-vis and NMR spectroscopy and mass spectrometry. A key goal of this work will be to distinguish products of beta-carotene antioxidant reactions, which consume beta-carotene and block radical chain reactions, from products of autoxidation reactions, which consume beta-carotene without blocking radical chain reactions. The validity of specific beta-carotene oxidation products as specific markers for beta-carotene antioxidant reactions will be tested in liposomes and biological membranes. Formation of putative antioxidant-specific marker products will be correlated with inhibition of specific biochemical markers for oxidative damage. Once the identities and applicability of antioxidant-specific marker products has been established, specific and sensitive high performance liquid chromatography, gas chromatography, or gas chromatography-mass spectrometry assays will be developed to measure product formation in biological samples. These studies then will be extended to C3H/10T1/2 and JB6 cell models for chemical carcinogenesis, in which carcinogen- and tumor promoter-induced neoplastic transformation in vitro is inhibited by beta-carotene or other antioxidants. beta-Carotene antioxidant reactions will be measured by analyzing marker products and correlating their formation with inhibition of oxidative cellular damage and transformation. The fundamental information generated by this projected about mechanisms of carotenoid anticarcinogenic actions can be applied to future investigations of cancer prevention in animals and humans.